Smooth muscle cell (SMC) proliferation plays a major role in vascular diseases. This cell cycle re-entry may involve modulation of cell cycle-regulatory protein expression. The SV40 large T antigen (TAg) is a viral oncoprotein able to transform cells in part due to its ability to complex with cellular proteins which behave as negative growth regulators; this suggested it expression in vascular smooth muscle cells as an approach to capture and identify negative regulators of smooth muscle cell cycling. Transgenic mice targeting a temperature-sensitive mutant SV40 TAg (tsA58) to SMC have been generated and have served as a source of SMC populations, which have in turn been the subject of initial studies with regard to cell cycle, differentiation, and the expression of TAg-associated proteins. The objectives of this proposal are the isolation of clonal populations of smooth muscle cells from adult and embryonic mice which are reversibly transformed by the association of the tsA58-TAg with a spectrum of negative growth regulatory proteins; and the use of these cells to study their range of phenotypes in relation to the spatiotemporal origin of the cells. The specific aims are: 1.) Isolation and characterization of clonal vascular SMC lines of neurocrest and mesodermal origin rom adult mice expression the tsA58-TAg antigen in smooth muscle cells; 2.) Isolation and characterization of clonal vascular smooth muscle cell lines from these progenitors from mice at various developmental stages; and 3.) Characterization of negative cell cycle regulatory proteins associating with T-antigen in adult and embryonic vascular smooth cells in vitro, and expressed during normal development of vascular smooth muscle tissue in vivo. These transgenic SMC exhibit a relatively differentiated phenotype in culture; and some isolates have shown temperature-dependent differentiation. This supports the concept that clonal SMC lines derived from adult and embryonic mice will provide a range of phenotypes for study. Proteins typical of adult as well as earlier developmental stages will be detected by co-immunoprecipitation with TAg. Initial studies have detected three phosphoproteins of unknown identity (pp130, pp160, pp170) associated with TAg in SMC. These and other such proteins will be evaluated for identity with known cell cycle regulatory proteins. Experiments will then be extended to assess the in vivo expression of negative growth regulators. These studies are expected to yield molecular information concerning the development spectrum of proteins regulating cycling within vascular SMC.